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Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.

Verdaguer J, Schmidt D, Amrani A, Anderson B, Averill N, Santamaria P - J. Exp. Med. (1997)

Bottom Line: The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined.Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities.These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Infectious Diseases, The University of Calgary, Faculty of Medicine, Alberta, Canada.

ABSTRACT
It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

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Responsiveness and peripheral frequency of beta cell–specific  CD8+ T cells in 8.3-NOD mice. (A) Proliferation of splenic CD8+ T  cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice to islet  cells. 2 × 104 splenic CD8+ T cells were incubated with γ-irradiated islet  cells for 3 d, pulsed with [3H]thymidine, harvested, and counted. Bars  show the standard error of the means. (B) Peripheral frequency of beta  cell–reactive CD8+ T cells in 8.3-NOD and 8.3–TCR-β–transgenic  NOD mice. 12 replicate cultures of serial dilutions of splenocytes (101– 105 cells/well) were stimulated with irradiated NOD islets (8/well) for 4 d,  expanded in rIL-2 (0.5 U/ml) for 10 d and restimulated once with islets  and rIL-2. The cultures were then challenged with 104 NIT-1 or L929-Kd  cells for 24 h, and the supernatants collected to measure their TNF-α  content. Cultures that secreted TNF-α in response to NIT-1, but not  L929-Kd, cells were considered to contain beta cell–reactive CD8+ T cells.  (C) General proliferative activity of splenic CD8+ T cells of 8.3-NOD  and 8.3–TCR-β–transgenic NOD mice. 2 × 104 splenic CD8+ T cells  were incubated with 10-fold serial dilutions of plate-bound KJ16 in  rIL-2–containing CM for 3 d, pulsed with [3H]thymidine, harvested, and  counted.
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Figure 2: Responsiveness and peripheral frequency of beta cell–specific CD8+ T cells in 8.3-NOD mice. (A) Proliferation of splenic CD8+ T cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice to islet cells. 2 × 104 splenic CD8+ T cells were incubated with γ-irradiated islet cells for 3 d, pulsed with [3H]thymidine, harvested, and counted. Bars show the standard error of the means. (B) Peripheral frequency of beta cell–reactive CD8+ T cells in 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 12 replicate cultures of serial dilutions of splenocytes (101– 105 cells/well) were stimulated with irradiated NOD islets (8/well) for 4 d, expanded in rIL-2 (0.5 U/ml) for 10 d and restimulated once with islets and rIL-2. The cultures were then challenged with 104 NIT-1 or L929-Kd cells for 24 h, and the supernatants collected to measure their TNF-α content. Cultures that secreted TNF-α in response to NIT-1, but not L929-Kd, cells were considered to contain beta cell–reactive CD8+ T cells. (C) General proliferative activity of splenic CD8+ T cells of 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 2 × 104 splenic CD8+ T cells were incubated with 10-fold serial dilutions of plate-bound KJ16 in rIL-2–containing CM for 3 d, pulsed with [3H]thymidine, harvested, and counted.

Mentions: To investigate whether peripheral T cells displaying the 8.3-TCR were functionally responsive to beta cells, we compared the proliferative activity of CD4+ T cell–depleted splenic CD8+ T cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice in response to irradiated NOD islet cells. As shown in Fig. 2 A, CD8+ T cells from 8.3-NOD, but not 8.3–TCR-β–transgenic NOD mice, proliferated vigorously in these assays, both in the presence and absence of exogenous rIL-2. The increased proliferative activity of splenic CD8+ T cells from 8.3-NOD versus 8.3–TCR-β–transgenic NOD mice resulted from an increased frequency of beta cell–reactive CD8+ T cells (measured at ∼1/17 versus 1/4,000 splenic CD8+ T cells, respectively; Fig. 2 B), rather than from differences in their general proliferative activity, since CD8+ T cells from both types of mice proliferated equally well in response to a plate-bound anti-Vβ8.1/8.2 mAb (KJ16; Fig. 2 C). Therefore, 8.3-NOD mice export numerous beta cell–specific CD8+ T cells to the periphery, and these autoreactive CD8+ T cells are not tolerant to antigen stimulation in vitro.


Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.

Verdaguer J, Schmidt D, Amrani A, Anderson B, Averill N, Santamaria P - J. Exp. Med. (1997)

Responsiveness and peripheral frequency of beta cell–specific  CD8+ T cells in 8.3-NOD mice. (A) Proliferation of splenic CD8+ T  cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice to islet  cells. 2 × 104 splenic CD8+ T cells were incubated with γ-irradiated islet  cells for 3 d, pulsed with [3H]thymidine, harvested, and counted. Bars  show the standard error of the means. (B) Peripheral frequency of beta  cell–reactive CD8+ T cells in 8.3-NOD and 8.3–TCR-β–transgenic  NOD mice. 12 replicate cultures of serial dilutions of splenocytes (101– 105 cells/well) were stimulated with irradiated NOD islets (8/well) for 4 d,  expanded in rIL-2 (0.5 U/ml) for 10 d and restimulated once with islets  and rIL-2. The cultures were then challenged with 104 NIT-1 or L929-Kd  cells for 24 h, and the supernatants collected to measure their TNF-α  content. Cultures that secreted TNF-α in response to NIT-1, but not  L929-Kd, cells were considered to contain beta cell–reactive CD8+ T cells.  (C) General proliferative activity of splenic CD8+ T cells of 8.3-NOD  and 8.3–TCR-β–transgenic NOD mice. 2 × 104 splenic CD8+ T cells  were incubated with 10-fold serial dilutions of plate-bound KJ16 in  rIL-2–containing CM for 3 d, pulsed with [3H]thymidine, harvested, and  counted.
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Related In: Results  -  Collection

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Figure 2: Responsiveness and peripheral frequency of beta cell–specific CD8+ T cells in 8.3-NOD mice. (A) Proliferation of splenic CD8+ T cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice to islet cells. 2 × 104 splenic CD8+ T cells were incubated with γ-irradiated islet cells for 3 d, pulsed with [3H]thymidine, harvested, and counted. Bars show the standard error of the means. (B) Peripheral frequency of beta cell–reactive CD8+ T cells in 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 12 replicate cultures of serial dilutions of splenocytes (101– 105 cells/well) were stimulated with irradiated NOD islets (8/well) for 4 d, expanded in rIL-2 (0.5 U/ml) for 10 d and restimulated once with islets and rIL-2. The cultures were then challenged with 104 NIT-1 or L929-Kd cells for 24 h, and the supernatants collected to measure their TNF-α content. Cultures that secreted TNF-α in response to NIT-1, but not L929-Kd, cells were considered to contain beta cell–reactive CD8+ T cells. (C) General proliferative activity of splenic CD8+ T cells of 8.3-NOD and 8.3–TCR-β–transgenic NOD mice. 2 × 104 splenic CD8+ T cells were incubated with 10-fold serial dilutions of plate-bound KJ16 in rIL-2–containing CM for 3 d, pulsed with [3H]thymidine, harvested, and counted.
Mentions: To investigate whether peripheral T cells displaying the 8.3-TCR were functionally responsive to beta cells, we compared the proliferative activity of CD4+ T cell–depleted splenic CD8+ T cells from 8.3-NOD and 8.3–TCR-β–transgenic NOD mice in response to irradiated NOD islet cells. As shown in Fig. 2 A, CD8+ T cells from 8.3-NOD, but not 8.3–TCR-β–transgenic NOD mice, proliferated vigorously in these assays, both in the presence and absence of exogenous rIL-2. The increased proliferative activity of splenic CD8+ T cells from 8.3-NOD versus 8.3–TCR-β–transgenic NOD mice resulted from an increased frequency of beta cell–reactive CD8+ T cells (measured at ∼1/17 versus 1/4,000 splenic CD8+ T cells, respectively; Fig. 2 B), rather than from differences in their general proliferative activity, since CD8+ T cells from both types of mice proliferated equally well in response to a plate-bound anti-Vβ8.1/8.2 mAb (KJ16; Fig. 2 C). Therefore, 8.3-NOD mice export numerous beta cell–specific CD8+ T cells to the periphery, and these autoreactive CD8+ T cells are not tolerant to antigen stimulation in vitro.

Bottom Line: The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined.Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities.These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Infectious Diseases, The University of Calgary, Faculty of Medicine, Alberta, Canada.

ABSTRACT
It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

Show MeSH
Related in: MedlinePlus